We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, alpha-naphthyl ester substrates are hydrolyzed by enzymatic action to alpha-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to alpha-naphthyl ester substrates, however, since beta-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.